Recently, the phenomenon of RNAi or double-stranded RNA (dsRNA)-mediated gene silencing has been recognized, whereby dsRNA complementary to a region of a target gene in a cell or organism inhibits expression of the target gene (see, e.g., WO 99/32619, published 1 Jul. 1999, Fire et al.; U.S. Pat. No. 6,506,559: “Genetic Inhibition by Double-Stranded RNA;” WO 00/63364: “Methods and Compositions for Inhibiting the Function of Polynucleotide Sequences,” Pachuk and Satishchandran; and U.S. Provisional Application Ser. No. 60/419,532, filed Oct. 18, 2002 (WO2004/035765, published 29 Apr. 2004: “Double-Stranded RNA Structures and Constructs, and Methods for Generating and Using the Same”). Double-stranded RNA (dsRNA) gene silencing presents a particularly exciting potential application for nucleic acid-based technology. Double-stranded RNA has been shown to induce gene silencing in a  number of different organisms. Gene silencing can occur through various mechanisms, one of which is post-transcriptional gene silencing (PTGS). In post-transcriptional gene silencing, transcription of the target locus is not affected, but the RNA half-life is decreased. Exogenous dsRNA has been shown to act as a potent inducer of PTGS in plants and animals, including nematodes, trypanosomes, insects, and mammals. Transcriptional gene silencing (TGS) is another mechanism by which gene expression can be regulated. In TGS, transcription of a gene is inhibited. In PTGS, the cytoplasmic compartment of the cell is the location of the machinery of the silencing complex. In TGS, the effector dsRNA is ostensibly active in the nuclear compartment of the cell. The potential to harness dsRNA mediated gene silencing for research, therapeutic, and prophylactic indications is enormous. The exquisite sequence specificity of target mRNA degradation and the systemic properties associated with PTGS make this phenomenon ideal for functional genomics and drug development.
Some current methods for using dsRNA in vertebrate cells to silence genes result in undesirable non-specific cytotoxicity or cell death due to dsRNA-mediated stress responses, including the interferon response. Early reports stated categorically that dsRNA-mediated toxicity in vertebrates is associated only with dsRNAs greater than 30 bps in length, not with siRNAs (short synthetic duplex dsRNA molecules of 21-23 bp) or other duplex dsRNAs of less than 30 bps. Despite the early acceptance of this dogma, more recent reports are establishing that non-specific silencing effects and other toxicity, including induction of cellular stress response pathways such as the interferon response, also occur with exogenously introduced siRNA molecules. Applicants, however, have demonstrated that intracellular expression of dsRNA molecules from a nucleic acid expression construct, including the long dsRNAs reported to induce toxicity in vertebrate cells, can be accomplished under conditions which do not trigger dsRNA-mediated toxicity. See, e.g., Published U.S. Patent Application No. 2004/0152117, Satishchandran, Pachuk, and Giordano. Accordingly, both long and short dsRNA molecules, including dsRNA hairpin molecules, can be used in mammals and other vertebrates  without inducing an interferon or other dsRNA-mediated stress response, if the dsRNA molecules are expressed within the host cell rather than introduced exogenously into the cell. However, a challenge remains in that the practical implementation of such dsRNA methods requires the efficient intracellular production and delivery of dsRNA molecules from dsRNA expression constructs.
For RNAi applications as well as use of nucleic acids for other mechanisms of biological activity, it is frequently desirable to express a biologically active nucleic acid intracellularly from a nucleic acid expression construct. The effectiveness of such methods depends upon an ability to efficiently express the selected nucleic acid in the target host cell in a therapeutically relevant manner, e.g., in a biologically active, non-toxic form to the desired target cell or cells in vivo or in vitro, in effective amounts and duration in the desired subcellular location or location(s). This presents a particular challenge in cells which are difficult to transfect, e.g., primary cells, certain cell lines, e.g., K5625, a human leukemia cell line, and for in vivo applications. Thus, improved expression systems, expression constructs and methods are needed for intracellular expression of nucleic acids from nucleic acid expression constructs in eukaryotes. Desirably, these methods may be used to provide nucleic acids capable of achieving any of their varied biological functions, including production of a desired polypeptide and/or a desired RNA effector molecule, e.g., a ribozyme, antisense, triplex-forming, and/or dsRNA in in vitro samples, cell culture, and intact animals (e.g., vertebrates, such as mammals, including humans).
In the decades since the advent of biotechnology, a huge variety of vectors, expression constructs, and expression systems, including circular plasmids, linearized plasmids, cosmids, viral genomes, recombinant viral genomes, artificial chromosomes, etc., have been developed for use in prokaryotes and/or eukaryotes. Use of these expression systems in bacterial cell culture has made such recombinant proteins as interferon (alpha), interferon (beta), erythropoietin, factor VIII, human insulin, t-PA, and human growth hormone a standard part of the pharmaceutical armamentarium. 
Among the tremendous variety of expression vectors and expression systems that have been developed in the field of biotechnology and molecular biology are expression systems containing multiple promoters on the same vector. One such type of multiple promoter expression system utilizes vectors containing multiple promoters (i.e., two or more promoters) that are active in a prokaryote or in the same subcellular compartment of a eukaryotic cell. For example, such multiple promoter systems in the art have been developed to permit expression of more than one sequence in the same compartment of the same cell (e.g., two distinct sequences or a sense and antisense sequence designed to form a dsRNA), or they may be used to express the same sequence within different cells or organisms (e.g., a prokaryote and a eukaryote) or to obtain more efficient transcription of a single operably linked sequence. Frequently seen are, e.g., multiple RNA polymerase II promoters or bacteriophage promoters on the same plasmid, such as, e.g., two polymerase II promoters-such as CMV and SV40, or a bacteriophage T7 promoter and a bacteriophage SP6 promoter.
Further, such multiple promoters can be arranged within the vector in any number of orientations and configurations. For example, two promoters can direct transcription both from the same or from the opposite strands of the vector. If oriented on the same strand, they drive transcription in the same direction within the vector. Alternatively, multiple promoters may be encoded on opposite strands and arranged either convergently or divergently with respect to each other in the same vector, in which case, transcription proceeds in opposite directions within the vector. Further, a variety of terms have been developed in the art to describe the relative position of multiple promoters within a single vector. The term “tandem” has been used to describe multiple promoters that all reside on, and are all operably linked to, the 5′ end of the sequence to be transcribed. Tandem promoters can be the same or different promoters. The term “flanking” promoters describes the orientation of multiple promoters in which the sequence to be transcribed is flanked on both the 5′ and the 3′ end by a promoter in such a manner that each promoter, when transcriptionally active, is capable of transcribing one strand of the  sequence to be transcribed. The flanking promoters can be the same or different promoters. E.g., a set of bacteriophage T7 RNA polymerase promoters flanking the 5′ and 3′ ends of a sequence is a common method for expressing separate sense and antisense strands to form duplex dsRNA (WO99/32619, Fire et al., published Jul. 1, 1999).
Multiple tandem promoters are described, e.g., in U.S. Pat. No. 5,547,862, which discloses a DNA vector which comprises an RNA transcription sequence positioned downstream from two or more tandem promoters which are recognized by distinct RNA polymerases and are each capable of promoting expression of the RNA transcription sequence.
A method for making mammalian collagen or procollagen in yeast is disclosed in U.S. Pat. No. 6,472,171 using a construct comprising, in opposite orientations, two mammalian collagen genes operably linked to a single or dual, divergent heterologous promoter(s). The promoter(s) driving the two collagen genes may be the same promoter, or different promoters, and may be used to provide for the coordinate, preferably simultaneous, expression of the two collagen genes.
Expression vectors containing dual bacterial promoters arranged in tandem and operably linked to a heterologous nucleic acid encoding a desired polypeptide are disclosed in U.S. Pat. No. 6,117,651. The dual promoter comprises a first component derived from a tac-related promoter (which is itself a combination of the lac and trp promoters) and a second promoter component obtained from a bacterial gene or operon that encodes an enzyme involved in galactose metabolism. The dual bacterial promoter system acts synergistically to provide a high level of transcription of the linked sequence in a prokaryotic cell such as E. coli. 
U.S. Pat. No. 5,874,242 discloses a vector which provides for the translation of an inserted coding sequence in both eukaryotic and prokaryotic host cells. Specifically, such vectors include either a bifunctional promoter (functional in both eukaryotes and prokaryotes) or dual promoters (promoters separately functional in eukaryotes and prokaryotes) for efficient expression in both prokaryotic and eukaryotic cells. 
There are a myriad of other examples in the art disclosing variations on themes of multiple promoters used in the same vector. However, there remains a need for new classes of expression vectors suitable for expression of multiple small RNA effector molecules, including multiple dsRNA hairpins of various lengths, e.g., shRNAs, bi- and multi-fingered dsRNA hairpins, long dsRNA hairpins, etc.), in a context useful for human therapeutic intervention. It is well-established that vector-directed expression short RNA effector molecules including short hairpin dsRNAs is most efficient when under the control of one of the mammalian promoter types which the cell naturally employs for expression of normally occurring small RNA molecules. These promoters typically comprise the family of RNA Polymerase III promoters. There are further defined in the literature three main subclasses of RNA polymerase III promoters, Type 1, Type 2 and Type 3. Prototypical examples of promoters in each class are found in genes encoding 5s RNA (Type 1), various transfer RNAs (Type 2) and U6 small nuclear RNA (Type 3). Another promoter family (transcribed by RNA Polymerase I) is also dedicated in the cell to transcription of small structural RNAs; however, this family appears to be less diverse in sequence than the RNA Polymerase III promoters. Finally, RNA Polymerase II promoters are used in the transcription of the protein-coding messenger RNA molecules, as distinguished from the small structural and regulatory RNA mentioned above. The majority of promoter systems known in the art utilize RNA Polymerase II promoters, which are not optimal for production of small RNAs.
RNA polymerase III promoter-based vectors containing one promoter have been described in the art (see, e.g., U.S. Pat. No. 5,624,803, Noonberg et al., “In vivo oligonucleotide generator, and methods of testing the binding affinity of triplex forming oligonucleotides derived therefrom”), and a description of U6-based vector systems can be found in Lee et al., Nat. Biotechnol. 20:500-05 (2002). Yu et al., Proc. Natl. Acad. Sci. USA 99:6047-52 (2002), describe an expression system for short duplex siRNAs comprising a T7 and U6 promoter. Miyagishi and Taira, Nat. Biotechnol. 20:497-500 (2002), describe expression plasmids for short duplex siRNAs comprising expression cassettes containing  tandem U6 promoters, each transcribing either the sense or the antisense strand of an siRNA, which are then annealed to form duplex siRNAs. Also described are expression plasmids including two such U6 based siRNA expression cassettes. The authors state that they utilized duplex siRNA expression cassettes because of their stability compared to stem-loop or hairpin-type dsRNA producing cassettes which include an unstable palindromic sequence.
In order to enable therapeutic utility of short RNA expression vector systems several criteria must be met with regard to structure as well as function. For human use, vectors must be designed to minimize the possibility of recombination events occurring both between vector sequences and genome sequences, and within the vector itself. These events are potentiated by regions of sequence identity (or homology) between the vector and the cellular genome sequence or between elements within the vector itself. The vectors of the present invention have been developed to provide for the use of multiple RNA expression cassettes, e.g., hairpin dsRNA, including shRNA as well as bi-finger and/or multi-finger dsRNA expression cassettes, from multiple promoters. The engineering of these multiple RNA pol III promoter vectors has been performed by selection of different promoter elements, together with spacing and orientation of expression cassettes within the recombinant expression vector to minimize the possibility of intra- or intermolecular recombination events. Assays to monitor the stability of such recombinant plasmids have been described previously (e.g. Focus 16:78, 1994, Gibco BRL Technical Bulletin (now Invitrogen Corp.)). Surprisingly, plasmid stability during bacterial fermentation and eukaryotic expression has been achieved despite the presence of multiple copies of palindromic or hairpin sequences, e.g., found in multiple (e.g., 2, 3, 4, 5) hairpin dsRNAs, as well as multiple copies of a polymerase III promoter sequence, e.g., 7SK and variants thereof, in the same or reverse orientation.
The multiple-promoter aspect of this invention in which each promoter controls expression of an independent RNA expression cassette, e.g., a shRNA expression cassette, is a critical feature in the design of, e.g., antiviral therapeutics, due to the nature of viral variation both within  human populations and temporally within a host due to mutation events. For example, this aspect of the invention provides a means for delivering a multi-drug regimen comprising several different dsRNA viral inhibitor molecules to a cell or tissue of a host vertebrate organism, such that the level of viral inhibition is potentiated and the probability of multiple independent mutational events arising in the virus and rendering dsRNA inhibition of viral replication ineffective, would be infinitesimally small.